The complement System of Plasma Proteins
Complement (C) is a cascading system of circulating blood plasma proteins. The C system consists of 20 different proteins, inclusive of the naturally occurring modulators of C activity, which collectively represent approximately 15% of the globulin fraction of normal human serum, H. J. Muller-Eberhard, Ann. Rev. Biochem. 44:697-725 (1975); K. B. Reid and R. R. Porter, Ann. Rev. Biochem. 50:433-464 (1981). In general C components are present in circulating blood plasma as non-activated native proteins. Complement components are activated sequentially upon the conversion of an inactive zymogen to an active proteolytic enzyme capable of cleaving, and thereby activating the next component in the reaction sequence. Polypeptide fragments of C proteins produced as tne result of cleavage by C enzymes, as well as subcomponents of the C1 complex, are designated by a small letter, e.g., the third component of C (C3) is cleaved to C3a and C3b -polypeptides as the result of C activation, Bull. W.H.O. 39:935-938 (1968); Bull. W.H.O. 59:489-491 (1981).
C activation can be initiated or triggered via two distinct and separate pathways termed the classical and alternative C activation pathways, H. J. Muller-Eberhard, Ann. Rev. Biochem. 44:697-725 (1975); M. K. Pangburn and H. J. Muller-Eberhard, Springer Semin. Immunopath. 7:163-192 (1984). The proteins unique to the classical C pathway are C1, C4 and C2. The C1 component is present in circulating blood plasma as a calcium dependent complex of subcomponents Clq, Clr and Cls which are present in the C1 complex in molar ratios of 1:2:2, respectively, N. R. Cooper, Adv. Immunol. 37:151-216 (1985). Classical C pathway activation is triggered upon binding of the Clq subcomponent of Cl to IgG or IgM immunoglobulin containing antigen-antibody complexes (immune complexes) or to a wide variety of biologically relevant activating substances, N. R. Cooper, Adv. lmmunol. 37:151-216 (1985).
Complement proteins unique to the alternative C activation pathway are Factors B, D and P (Properdin). However, C3 and the complement regulatory proteins Factor H and Factor I, although not unique to the alternative pathway, are usually considered to be alternative pathway components since they are required for full functional alternative pathway activation to occur, R. D. Schreiber, et al., Proc. Natl. Acad. Sci. (USA) 75:3948-3952 (1978); M. K. Pangburn and H. J. Muller-Eberhard, Springer Semin. Immunopath. 7:163-192 (1984). Alternative C pathway activation can be triggered by IgA-immunoglobulin containing immune complexes, or a variety of fungal, viral, parasitic or gram negative bacterial surface determinants.
C4 activation is a key reaction step in triggering the classical C pathway. The C4 present in circulating blood plasma is a 200,000 molecular weight (MW) protein comprised of three disulfide bonded subunits, .alpha.-93,000 MW, .beta.-75,000 MW, .gamma.-32,000 MW, B. F. Tack et al., Meth. Enzymol. 80:64-101 (1980). The .alpha.-subunit of native C4 contains an intra-chain, internal, active thiol ester bond which is normally shielded or inaccessible to nucleophilic attack by solvent water molecules. C4 is activated by the C1s proteolytic enzyme. C1s cleaves the C4 .alpha.-chain at peptide bond 77 resulting in the production of C4a and C4b fragments. The C4a fragment, a 9000 MW peptide, is one of the C anaphylatoxins, J. P. Gorski, et al., J. Biol. Chem. 256:2707-2711 (1981). Cleavage of the C4 .alpha.-chain by C1s, with resultant release of the C4a peptide, exposes the internal, active thiol ester bond present in the larger C4b fragment to nucleophilic attack by target surface acceptor molecules (amino or hydroxyl chemical groups) or by solvent water molecules. The successful nucleophilic attack by a target acceptor molecule, which must occur within milliseconds after C4 .alpha.-chain cleavage by C1s, results in the formation of a covalent ester bond between the C4b fragment and the target surface, J. Janatova and B. F. Tack, Biochem. 20:2394-2402 (1981); R. A. Harrison, et al., Proc. Natl. Acad. Sci. (USA) 78:7388-7392 (1981). Approximately 10% of nascent C4b fragments will bind to target acceptor molecules While the remaining 90% will react with the hydroxyl chemical group of solvent water. The C4b fragments which have reacted with water are unable to subsequently bind to target surface acceptors and accumulate in the fluid phase reaction solution as inactive by-products of the classical pathway activation event.
Both target acceptor bound and fluid-phase C4b fragments are subject to further fragmentation and degradation reactions as the result of normal physiological control mechanisms. Thus, the C4b .alpha.-chain of surface-bound and fluid-phase C4b is degraded to C4c and C4d fragments by the naturally occurring C regulatory proteins C4 Binding Protein (C4BP) and Factor I. C4BP acts as a required cofactor which must bind to the C4b fragment before Factor I mediated cleavage can occur, T. Fujita, et al., J. Exp. Med. 148:1044-1051 (1978); B. Dahlback and B. Hildebrand, Biochem. J. 209:857-863 (1983). C4c and C4d fragments have molecular weights of 146,000 and 45,000, respectively. The C4d fragment is derived from the portion of the C4 .alpha.-chain containing the active thiol ester site.
Activation of C3 is the first reaction step shared by both C pathways. Thus, not only is C3 the most abundant protein of the C system (the normal human plasma concentration of C3 is 1200 .mu.g/mL), it is also a very centrally important component in the C activation sequence. The C3 convertase enzymes of either C pathway, i.e., the C4b,2a enzyme of the classical or the C3b,Bb enzyme of the alternative pathway, cleaves the C3 .alpha.-chain at peptide bond 77 resulting in the production of C3a and C3b fragments. The C3a fragment is one of the C anaphylatoxins, T. E. Hugli, Contemp. Topics Molec. Immunol. 7:181-214 (1978). The .alpha.-chain of the larger C3b fragment also contains an intra-chain, internal, active thiol ester bond which becomes accessible to nucleophilic attack by target surface acceptor molecules or by solvent water molecules. The successful nucleophilic attack by a target acceptor molecule, which must occur within milliseconds after C3 .alpha.-chain cleavage, results in the formation of a covalent ester bond between the C3b fragment and the target surface, J. Janatova, et al., Biochem. 19:4479-4485 (1980). Analogous to C4, approximately 10% of nascent C3b fragments will bind to target acceptors while the remaining 90% react with solvent water molecules. The C3b fragments which have reacted with water are unable to subsequently bind to target surface acceptors and they therefore accumulate in the fluid phase reaction solution as inactive by-products of any C activating event.
As part of normal physiological control mechanisms, both acceptor bound and fluid phase C3b fragments are subject to further fragmentation and degradation reactions. Thus, the C3b .alpha.-chain of surface bound or fluid phase C3b is degraded to iC3b and C3f fragments by the naturally occurring C regulator proteins, Factors H and I. Factor H acts as a required cofactor which must bind to the C3b fragment before Factor I mediated cleavage can occur, K. Whaley and S. Ruddy, J. Exp. Med. 144:1147-1163 (1976); M. K. Pangburn, et al., J. Exp. Med. 146:257-270 (1977); R. A. Harrison and P. J. Lachmann, Molec. Immunol. 17:9-20 (1980); G. D. Ross, et al., J. Immunol. 129:205-2060 (1982). The iC3b fragments can be further degraded by a variety of proteolytic enzymes, e.g., Factor I in the presence of the CRI C receptor, trypsin, elastase or plasmin, resulting in the production of C3c and C3d,g (.alpha..sub.2 D) fragments, D. T. Fearon and W. W. Wong, Ann. Rev. Immunol. 1243-271 (1983). The C3d,g (.alpha..sub.2 D) fragment appears to be the final C3 degradation fragment normally produced in circulating blood plasma, C. D. West, et al., J. Clin. Invest. 46:539-548 (1967); A. E. Davis, et al., J. Immunol. 132:1960-1966 (1984). However, C3d,g can be further degraded in extravascular sites of inflammation by a variety of proteolytic enzymes to yield C3d and C3g fragments. The molecular weights of the various physiological degradation fragments of C3 are: iC3b-185,000; C3f-2300; C3c-145,000, C3d,g-40,000; C3d-30,000, C3g-10,000.
Activation of either C pathway through the C3 step results in assembly of C5 activating enzymes, also termed C5 convertase enzymes. The classical and alternative pathway C5 convertase enzymes are C4b,2a,3b and C3b,Bb,C3b, respectively. Both C5 convertase enzymes cleave the C5 .alpha.-chain at peptide bond 74 resulting in the production of C5a and C5b fragments. The C5a fragment is one of the C anaphylatoxins, T. E. Hugli, CRC Crit. Rev. Immunol. 1:321-366 (1981). The larger C5b fragment remains bound to the C5 convertase enzyme which produced it, and upon interaction with C6 and C7 a C5b,6,7 complex is formed which becomes bound to the target surface, E. R. Podack, et al., J. Immunol. 121:484-490 (1978). The C5b,6,7 complex subsequently binds C8 and multiple C9 molecules leading to C5b-9 complex assembly. It is the assembled C5b-9 complex which is responsible for the irreversible target membrane surface damage associated with C activation, P. J. Lachmann and R. A. Thompson, J. Exp. Med. 131:643-657 (1970); O. Gotze and H. J. Muller-Eberhard, J. Exp. Med. 132:898-915 (1970); W. P. Kolb, et al., J. Exp. Med. 135:549-566 (1972). The C5b fragment, unlike C4b and C3b, does not contain an intra-chain thiol ester bond and is not degraded by Factor I nor any other known physiologically occurring plasma enzyme or inhibitor. However, plasma inhibitors do exist to protect bystander target surfaces from nascent C5b,6,7 complex binding and subsequent C5b-9 complex assembly. The major inhibitor of bystander cell membranolysis mediated by the terminal C components is the naturally occurring blood plasma protein termed S-protein, E. R. Podack, et al., J. Immunol. 120:1841-1848 (1978). Since S-protein binds at the C7 stage of terminal complex assembly, a significant percentage of C5 activation events result in the formation of SC5b,6,7 complexes in free solution. SC5b-7 complexes combine with C8 and multiple C9 molecules to produce a fluid-phase SC5b-9 complex as an inactive by-product of C5 through C9 terminal pathway activation, W. P. Kolb and H. J. Muller-Eberhard, J. Exp. Med. 141:724-735 (1975).